To localise cytokinins in <I>V. inaequalis</I> infected apple tissue immuno-fluorescence microscopy was employed and demonstrated that cytokinins localised around the <I>V. inaequalis</I> hyphae. PCR and Southern analysis were used in an attempt to identify the gene(s) involved in cytokinin production by <I>V. inaequalis</I>. Cytokinin production was also confirmed for the light leaf spot pathogen of brassicas.<I> Pyrenopeziza brassicae</I>, using HPLC-ELISA. Another plant pathogen, <I>Leptoxphaeria maculans</I>, appeared not to be producing cytokinins under the conditions tested. Cytokinin-O-glucoside cleaving β-glucosidase activity that released free base zeatin from zeatin-O-glucoside was identified for the first time in plant pathogenic fungi. In <I>V. inaequalis</I> cytokinin-O-glucoside cleaving β-glucosidase activity was found to significantly increase, relative to total β-glucosidase activity was found to significantly increase, relative to total β-glucosidase activity, when the fungus was grown under growth limiting instead of nutrient rich-conditions <I>in vitro</I>. A protocol was developed for the purification of the enzyme that produced this activity and it was purified to apparent homogeneity. Attempts were made to gain a handle on the gene(s) involved in producing this activity using heterologous Southern hybridisation to known cytokinin glucoside cleaving β-glucosidases from plants. In addition cytokinin receptors were sought from apple in order to provide a means to assess fungal cytokinin production <I>in planta</I>. Genes might be involved in the biotrophic phase of <I>V. inaequalis</I> infection were sought by constructing a genomic library of <I>V. inaequalis</I>. The library was differentially screened with 1<SUP>st</SUP> strand cDNAs derived from <I>V. inaequalis</I> grown under growth limiting and nutrient rich conditions to identify putative fungal genes expressed <I>in planta</I>. The findings of this study were related to the modes of nutrition of the plant pathogens studied. It emerged that, in general, cytokinin production and cytokinin-O-glucoside cleaving β-glucosidase activity were a feature of biotrophic and hemibiotrophic fungi but not necrotrophic fungi.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:597958 |
| Date | January 1999 |
| Creators | Cooper, S. J. |
| Publisher | University of Cambridge |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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